FIGURE 5.

Exonuclease III footprints of the PIC. A, the 5′-end of the non-template strand of HIS4(−93/+109) was labeled with ³²P to determine the downstream boundary. The labeled DNA (1.0 pmol) was incubated with TFIIB (2.0 pmol), TFIIA (1.9 pmol), TBP (1.0 pmol), TFIIE (2.4 pmol), and TFIIH (1.0 pmol) with or without the nucleotides as indicated above the lanes, followed by treatment with exonuclease III (ExoIII) and gel electrophoresis. The positions of protected fragments are indicated on the right, and the positions of molecular markers are indicated on the left. The assay was performed in the presence (left) or absence (right) of TFIIE and TFIIH*. B, to determine the upstream boundary, the 5′-end of the template strand of HIS4(−132/+109) was labeled with ³²P, incubated with the pol II and GTFs, and treated with 3′-exonuclease. The arrow indicates the upstream boundary of the DNA of the closed complex. The assay was performed in the presence (left) or absence (right) of TFIIE and TFIIH*.